The separation of biological molecules, such as proteins, peptides and nucleic acids, prior to or in parallel with their identification and quantification, can be achieved by a variety of techniques. Gel electrophoresis is a technique which is commonly used to separate these biological molecules on the basis of their size and/or their charge.
Mass spectrometry has today become the method of choice for the determination of the identity and composition of proteins and peptides. To allow collection of the information required a protein is in a first step cut up into peptides by either enzymatic or chemical means. The most common approach is enzymatic digestion using enzyme(s) which cut the protein at specific amino acid residues, a typical example being trypsin which hydrolyses the protein after lysine or arginine residues. It is, when tryptic digestion is carried out on a sample containing a very limited number of proteins, possible to determine the identity of the protein present from the masses of the peptides resulting from the digestion. A second approach used for identification purposes is the generation of a collision induced secondary mass spectra ion from ions separated in a primary mass spectrum. As the secondary mass spectra contains information on the masses of the amino acid residues constituting a peptide, these masses in combination with the mass of the ion selected in the primary spectrum can be used for identification of the tryptic peptide and the protein corresponding to this peptide. Evidently MS/MS spectra can be used not only for the identification and characterisation of enzymatically digested peptides, but also for peptides originally present in the biological sample. In proteomic studies it is common to use MS or MS/MS not only for identification of protein but also for relative quantification (Aebersold et al; Nature, 2003, 422, 198-207).
A sample applied to a MALDI-MS target is only allowed to contain a limited number of peptides and similarly ESI-MS can only accept a limited number of peptides per time unit. The sample is normally a very complex mixture containing many thousand of proteins which after digestion could easily correspond to one hundred thousand to more than one million peptides. There is therefore a need for rigorous separation of the peptides prior to MS characterisation and quantification. A variety of different separation methods including electrophoretic and chromatographic methods can be used; normally multiple separation steps are required.
Separation can be conducted solely at the protein level prior to tryptic digestion. A typical example of this approach is two-dimensional (2-D) electrophoresis. Alternatively, separation can be carried out at the protein level in the first step, followed by digestion and finally separation of the resulting peptides prior to MS. One example of this approach uses reverse phase chromatography (RPC) at the protein level followed by digestion and reverse phase chromatography separation of resulting peptides prior to ESI MS/MS. Another approach described is SDS-electrophoresis at the protein level followed by digestion and RPC (Breci et al; Proteomics, 2005, 5, 2018-2028). Finally tryptic digestion can be carried out prior to multidimensional separation at the peptide level. Approaches of this type include MudPit (Washburn et al; Nat Biotechnol., 2001, 19, 242-247), more conventional ion-exchange chromatography followed by RPC (Peng et al; Journal of Proteome Research, 2003, 2, 43-50) as well as peptide isoelectric focusing (IEF) followed by RPC (Cargile et al; Electrophoresis, 2004, 25, 936-945).
When tryptic digestion is the first step, an alternative approach is to decrease the complexity of the sample by the use of methods which allow the selection of a small fraction of the peptides (e.g. iCAT [Aebersold et al; Proteomics, 2005, 5, 380-387] alt COFRADIC [Vandekerckhove et al; Nat Biotechnol., 2003, 21, 566-569]).
Generally electrophoretic techniques like IEF and SDS electrophoresis give, when used at the protein level in gel, much better resolution and protein yields than chromatographic alternatives. 2-D electrophoresis based on the combination of these two techniques, IEF and SDS, is also a commonly used approach when separation of very complex samples is conducted at the protein level. The disadvantages with electrophoretic techniques are however that they are labour intensive, often demand craftsmanship and that they are hard to automate.
Problems can also be encountered extracting the analyte from the gel.
The processing of gel fractions containing peptides, proteins, carbohydrates or nucleic acids from electrophoretic gels in order to facilitate further separation or to enable analyte analysis presents significant difficulties to the operator. Where the gel is present on a glass or plastic plate, individual bands or fractions must be blotted or scraped from the plate, typically with a spatula or sharp knife, and carefully transferred either to a second gel or a reaction vessel for further analysis. In the situation where the gel is supported on a plastic sheet, as with an IPG strip, the strip must be carefully cut with scissors or a sharp blade into a series of pieces which can then be transferred to another gel or reaction vessel for further processing/analysis.
Automatic sampling systems are known for removing bands or spots from gels, such as those described in WO 02/071072. In fact, 2-D electrophoresis frequently employs automatic spot pickers in which gels are generally stained to detect the protein or peptide samples. However, these systems usually involve aspiration of the gel into a pipette which leads to losses due to gel sticking to the outside or inside of the pipette. Furthermore, these systems are labour intensive and time consuming, involving protein/peptide staining and careful use of the apparatus to avoid losses and contamination.
It will be understood by the skilled person that the process of removing bands or fractions of gel manually from a plate or strip is time consuming as painstaking care must be taken in order to ensure that the gel is divided evenly into the appropriate number of fractions, that there is quantitative recovery of the analyte from the gel, and that cross-contamination from ‘dirty’ instruments used in the transfer process is avoided. The problem of cross-contamination is particularly significant where the analyte has been separated using IPG strips and scissors or a scalpel is used to cut the strip into bands for further processing/analysis, as the blades of these instruments must be thoroughly cleaned before the next band of gel is excised from the strip. Furthermore, such processes generally involve the additional step of pre-staining the gel in order to detect peptides or proteins, such systems are extremely labour intensive.
It will also be understood by the skilled person that the problems described above experienced in removing and transferring gel bands from a plate or IPG strip to a second gel or reaction vessel for further processing will be exacerbated with an increasing number of bands or fractions. Thus, for example, where an IPG strip has to be divided into some 50 pieces and each of the 50 pieces transferred to another gel or a reaction vessel, there is an increasing likelihood of cross-contamination and poor recoveries.
To avoid the problem with sample extraction from gels, isoelectric focusing separation can be carried out in liquid phase (Zuo et al.; Methods Mol Biol., 2004, 244, 361-75). The equipment used by Zuo et al. comprises a series of chambers separated by membranes titrated to specific pH-values. However, one disadvantage of this approach is that peptides and proteins have low solubility in the vicinity of their isoelectric points; the resulting precipitation and aggregation can lead to problems of poor resolution of the peptides and proteins during the isoelectric focusing.
Michel et al. (Electrophoresis, 2003, 24, 3-11) describe a technique which allows the fractionation of complex biological samples according to their isoelectric point (pl) as well as the direct recovery of the compounds for further analysis. The technique, termed ‘off-gel IEF’, involves dividing IPG strips into a series of wells using a multiwell device which is open at both ends, adding protein sample in an IPG buffer and then conducting electrophoresis to separate the protein mixture. The content of each well is then removed for protein analysis by mass spectrometry and the technique shown to effect a resolution of 0.1 pH units. However, as in the approach of Zuo et al. discussed above, the proteins are present in liquid phase during focusing which increases the risk of precipitation and aggregation. With the geometry resulting from the approach of Michel et al., the proteins will be present in a region with much lower electric field than would be the case if the focusing was done solely in the gel in the absence of any solution added in the multiwell device. Compared to conventional gel focusing the result is lower resolution and a demand for longer focusing times.
The same group (Heller et al.; Electrophoresis, 2005, 26, 1174-1188) has recently reported the use of ‘off-gel IEF’ for the separation and identification of proteins and their isoforms by use of a two-stage process, the first involving separation of the proteins and their isoforms on the basis of their pl's and the second the separation and identification of the trypsinized peptide fragments.
IEF can also be carried out in configurations where separated proteins are collected in solution in chambers separated with membranes (Righetti et al; J. Biochem. Biophys. Meth., 1987, 15, 199-206). This approach is also limited by the fact that proteins dose to their isoelectric point tend to aggregate and precipitate.
Other systems have been disclosed which describe methods for processing proteins in gels wherein gel fragments containing proteins are isolated from the gel, subjected to proteolytic digestion and then the cleavage peptides produced are identified. Such an automated system is described in WO 02/071072, in which isolated protein-gel fragments are directly transferred to a corresponding number of reaction vessels of a first microtitre plate by a robotic arm device, the base of the microtitre plate having a hydrophobic filter membrane, and incubated with a protease. Following hydrolysis, the peptide products are filtered through the hydrophobic filter membrane into a second microtitre plate and concentrated for subsequent analysis.
Thus electrophoretic separation in gel provides outstanding resolution but, as discussed above, often involves problems with sample transfer from the gel to liquid phase and is difficult to automate.
It is therefore an object of the present invention to provide methods and systems which facilitate the preparation of gel fractions and enable the further processing and manipulation thereof while ameliorating the problems encountered in the prior art. Another object of the invention is to provide such methods and systems without the need to pre-stain gels for the detection of such analytes. A further object of the present invention is to provide methods and systems for adding reagents to gel fractions and for eluting analyte, either prior to or following chemical or enzymatic modification, from a gel.